Inhibitors of metazoan parasite proteases

ABSTRACT

Compositions and methods for treating a patient infected with a metazoan parasite by inhibiting the enzymatic action of the metazoan parasite protease. The compositions comprise at least one metazoan protease inhibitor which binds to the S2 subsite and at least one of the S1 and S1&#39; subsites of the metazoan parasite protease. The methods comprise administration to a patient infected with a metazoan parasite of at least one metazoan protease inhibitor in an amount effective to inhibit the protease of the metazoan parasite, thereby killing the parasite.

This invention was made with Government support under Contract No. MDA972-91-J-1013, awarded by DARPA (Department of Defense); and Grant No.890499 awarded by UNDP/World Bank/WHO Special Programme for Research andTraining in Tropical Diseases (TDR). The Government has certain fightsin this invention.

This patent application is based on PCT International ApplicationPCT/US93/08708 filed on Sep. 13, 1993 (corresponding to PCT publishedapplication WO 94/06280, published on Mar. 31, 1994) which is acontinuation-in-part of U.S. patent application Ser. No. 07/943,925filed on Sep. 11, 1992, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates generally to compositions and methods useful inthe treatment of certain infectious diseases. More specifically, theinvention relates to compositions which inhibit proteases, such asmalaria cysteine protease. Compounds that inhibit these proteases areuseful in the prevention and treatment of malaria, schistosomiasis andother infectious diseases.

Proteases are involved in many important biological processes includingprotein turnover, blood coagulation, complement activation, hormoneprocessing, and cancer cell invasion. Thus, they are frequently chosenas targets for drug design and discovery. The critical role certainproteases play in the life cycle of parasitic organisms also makes themattractive drug design targets for certain infectious diseases.

Schistosomiasis (bilharziasis) is a parasitic disease caused byschistosomes (blood flukes) that generally live in the veins of the gutand liver of a human host. Adult worms can survive up to 20 years.Female adult worms release thousands of eggs each day, which often findtheir way to tissues such as liver, brain, and lung, where they causeconsiderable damage by stimulating the body to form inflammation andscar tissue around them. Most eggs pass through the bladder or wall ofthe gut. Once outside, they hatch and infect water snails. The parasitemultiplies inside the snail, giving rise to thousands of cercariae thatexit the snail and swim free in search of a host in which to completetheir life cycle.

Malaria is another well known infectious disease caused by protozoa ofthe genus Plasmodium, which are transmitted by bites of infectedmosquitoes. Infection with Plasmodium falciparum, the most virulenthuman malarial pathogen, is estimated to be responsible for over 1million deaths each year. The most valuable of the heretofore developedclasses of antimalarial drugs are the quinoline-containing compounds,such as chloroquine and mefloquine; chloroquine has been especiallyeffective as both a preventative and a curative. A serious problem inthe treatment and control of malaria has been the increasing resistanceof populations of P. falciparum to these known antimalarial drugs. Inaddition, reports of multi-drug resistance makes the search for noveltherapies especially urgent. Thus, there remains a great need toidentify new compounds that have antimalarial capabilities.

During the trophozoite stage, the parasites infect red blood cells(erythrocytes) where they reproduce asexually. At the completion of eachasexual cycle, the red blood cells lyse and merozoites are releasedwhich invade new red blood cells. This cycle of lysis and re-infectionis responsible for the major clinical manifestations of malaria.

Most anti-malarials are blood schizontocides which are active againstthe parasites during the intra-erythrocytic stage of its life cycle.Sulphones and sulphonamides inhibit the synthesis of dihydrofolic acid,while biguanides and diaminopyrimidines inhibit the synthesis oftetrahydrofolic acid. Although the mechanism of these anti-malarials isknown to involve interference with the parasites' ability to synthesizenucleic acids [Bruce-Chwatt, L. J., Essential Malariology (Wiley, NewYork (1985))], the mechanism of action of the quinoline-containingcompounds has until recently been surprisingly elusive. Recent workprovides strong evidence that the quinoline derivatives work byinterfering with the detoxification activity of a heme polymerase[Slater and Cerami, Nature 355, 167 (1992)].

During the erythrocytic phase, the parasites degrade hemoglobin as aprimary cysteine protease involved in the degradation of hemoglobin, theparasites' primary source of amino acids [Rosenthal, P. J. et al., J.Clin. Invest. 82, 1560 (1988)]. Blocking this enzyrme with cysteineprotease inhibitors (such as E-64 and Z-Phe-Arg-FMK) in culture arrestsfurther growth and development of the parasites [Rosenthal, P. J. etal., Mol. Biochem. Parasitol. 35, 177 (1989)]. Because humans (and,probably, most other mammals) do not have an analogous hemoglobinase,inhibition of this protease (either alone or in conjunction withestablished therapies) provides an attractive strategy for the treatmentof malaria. Moreover, inhibition of analogous proteases present in othermetazoan parasites would similarly provide potentially valuabletechniques for treatment of human and animal patients infected withthose parasites.

It is an object of the present invention to provide compositions andmethods for treatment of malaria.

It is a further object of the present invention to provide compositionsand methods useful in the treatment of other infectious diseases causedby metazoan parasites.

SUMMARY OF THE INVENTION

In accordance with the present invention, there are providedcompositions and methods for treating a patient infected with a metazoanparasite by inhibiting the enzymatic action of the metazoan parasiteprotease. These compositions and methods have particular utility in thetreatment of schistosomiasis, malaria, and other infectious diseases.The compositions comprise at least one metazoan protease inhibitor (ashereinafter defined) containing specific structural elements which bindto the S2 subsite and at least one of the S1 and S1' subsites of themetazoan parasite protease. The protease inhibitors of the presentinvention generally include at least two homoaromatic or heteroaromaticring systems, each comprising one to three rings, joined together bysuitable linkers. The compositions of the present invention are useful,for example, to inhibit the action of trophozoite cysteine protease,thereby preventing degradation of hemoglobin, the primary source ofamino acids for the pathogen that causes human malaria. The methods ofthe present invention comprise administration to a patient infected witha metazoan parasite of at least one metazoan protease inhibitor in anamount effective to inhibit the protease of the metazoan parasite,thereby killing the parasite.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be better understood with reference to theaccompanying drawings, in which:

FIG. 1 is the IC₅₀ curve for oxalicbis-[(2-hydroxyl-1-naphthylmethylene)-hydrazide] against the malarialcysteine protease;

FIG. 2 illustrates the inhibition of the parasite uptake of ³H-hypoxanthine by oxalicbis-[(2-hydroxyl-1-naphthylmethylene)hydrazide];

FIG. 3 is a schematic of oxalicbis-[(2-hydroxy-1-naphthylmethylene)-hydrazide] docked into the activesite of trophozoite cysteine protease; and

FIG. 4 illustrates inhibition of the parasite's ability to invade redblood cells by oxalic bis-[(2-hydroxyl-1-naphthylmethylene)hydrazide].

DETAILED DESCRIPTION OF THE INVENTION

Pursuant to the present invention, compositions and methods forinhibiting the enzymatic action of metazoan parasite proteasescomprising an effective amount of at least one metazoan proteaseinhibitor (as hereinafter defined) are provided. These compositions haveutility in the prevention and treatment of schistosomiasis, malaria, andother infectious diseases. In the case of malaria, the compositions ofthe invention inhibit the trophozoite cysteine protease. Inschistosomiasis, the enzyme inhibited by the compositions of theinvention is the adult cysteine protease (or "hemoglobinase").

The inhibitors of the present invention have particular utility againstthe metazoan parasites Plasrnodium falciparum (which causes malaria),Schistosoma mansoni (which causes schistosomiasis), and Trypanosomacruzi (which causes chagas' disease). In addition, proteases specific tothe following metazoan parasites may also be inhibited by compositionsin accordance with the present invention: Giardia lamblia, Entoemebahistolytica, Cryptospiridiurn spp., Leishmania spp., Brugia spp.,Wuchereria spp., Onchocerca spp., Strongyloides spp., Coccidia,Haemanchus spp., Ostertagia spp., Trichomonas spp., Dirofilaria spp.,Toxocara spp., Naegleria spp., Pneumocystis carinii, Ascaris spp., otherTrypanosoma spp., other Schistosome spp., other Plasrnodium spp.,Babesia spp., Theileria spp., Anisakis and Isospora beli.

The structure for a model metazoan protease, malaria cysteine protease,was developed using sequence information and the x-ray structures ofpapain and actinidin as a basis for homology modeling following theapproach of Sutcliffe and co-workers [Sutcliffe. M. J. et al., ProteinEngineering 1, 377 (1987); Sutcliffe, M. J. et al., Protein Engineering1, 385 (1987)]. The protease model structure was used as target receptorin a search for potential ligands. DOCK3.0 is an automatic method toscreen small molecule databases for possible ligands of a given receptor[Meng, E. C. et al., J. Comp. Chem. 13, 505 (1992)]. DOCK3.0 was used tosearch the Fine Chemicals Directory of approximately 56,000 commerciallyavailable small molecules. The 2200 molecules with the best shapecomplementarity scores and the 2200 with the best force field scores(estimated interaction energies) were saved. The resulting 4400compounds were visually screened in the context of the active site usingthe molecular display software MidasPlus [Ferrin, T. et al., J. Mol.Graphics 6, 13 (1988)]. An effort was made to choose a diverse group ofcompounds from these lists. Thirty-one compounds were ultimately chosenfor initial testing on the malaria cysteine protease, and from thesecompounds a number of potential inhibitors were identified fromexperimental tests against the enzyme system. This constituted the firstsuccessful attempt to use computer-assisted docking of non-peptidicmolecules to a model of an enzyme's three-dimensional structure.

Of the compounds originally considered as potentially useful inhibitors,oxalic bis-[(2-hydroxyl-1-naphthylmethylene)-hydrazide] andN,N-bis(2-hydroxy-1-naphthylmethylene)hydrazone were identified as thebest inhibitors of the protease. The IC₅₀ ofN,N-bis(2-hydroxy-1-naphthylmethylene)hydrazone for enzyme inhibitionagainst the substrate Z-Phe-Arg-AMC was 5 μM. The IC₅₀ of oxalicbis[(2-hydroxy-1-naphthylmethylene) hydrazide] in the same assay was 6μM (FIG. 1); more importantly, the compound kills parasites in cultureat approximately the same concentration as judged by hypoxanthineuptake, a standard measure of Plasmodium viability (FIG. 2). Oxalicbis[(2-hydroxy-1-naphthylmethylene)hydrazide] was selected as the basisfor further structure/activity studies and was designated as compound143A.

It had previously been demonstrated that papain-like cysteine proteasescontain active sites that could accommodate up to seven amino acids[Berger & Schechter, Phil. Trans. Roy Soc. Ser. B 257, 249 (1970)]. Inthe literature nomenclature, the active sites are numbered consecutivelystarting from the catalytic site S1 to Sn towards the amino terminus andS1' to Sn'0 towards the carboxyl terminus. The S2, S1 and S1' subsitesare believed to be the most important for binding.

Because oxalic bis-[(2-hydroxyl-1-naphthylmethylene)-hydrazide] is arigid and symmetric molecule, there are essentially two ways to orientthe compound in the active site. In both, the compound lays across theactive site cleft of malaria cysteine protease with one naphthol groupfitting into the S2 specificity pocket and the other stacking with theindole ring of tryptophan 177 in the S1' pocket. The presumed bindingmode is shown in FIG. 3. This orientation was chosen for illustration,as it maximizes the compound's interaction with the enzyme, eachhydroxyl being within hydrogen bonding distance to a suitable residue inthe enzyme (Ser160 and Gln19, respectively). Alternatively, the compoundcould be rotated 180° through the horizontal axis; although thehydroxyls do not interact with the enzyme, they presumably interact withsolvent water molecules.

The original ligand coordinates of oxalicbis-[(2-hydroxyl-1-naphthylmethylene)hydrazide] supplied by DOCK3.0 wereoptimized by rotation and translation of the ligands without internalchanges in ligand bond length, bond angle or torsional angles subject tothe constraints of a semi-empirical molecular force field to provide arigid body minimized complex for the molecule. Starting with the rigidbody minimized complex of oxalicbis-[(2-hydroxyl-1-naphthylmethylene)-hydrazide], the volume limits ofthe S2 and S1' subsites were then explored by varying the size,composition and substituent patterns on the aromatic ring systemsdetermined to be effective in binding to the target subsites of theprotease model. Simultaneously, the linker length was varied to find theoptimum spacing between the ring systems. In this manner, optimal ringsystems and spacer lengths have been determined in accordance with thepresent invention for protease inhibitors which bind to the S2 and S1'subsites.

In addition, it has further been determined in accordance with thepresent invention that compounds comprising aromatic ring systemscharacteristic of the inhibitors useful for binding to the S2 and S1'subsites but joined by shorter linkers than would be appropriate forbridging these subsites were also effective in inhibiting metazoanparasite proteases. Modeling studies with these shorter compoundsindicates that one of the aromatic ring systems binds to the S2 subsite,as is the case with the inhibitors based on oxalicbis-[(2-hydroxyl-1-naphthylmethylene)-hydrazide]. However, instead ofinteracting with the S1' subsite, the second aromatic ring system of theshorter compounds binds to the S1 subsite. These shorter compounds areof particular interest as they can be synthesized in a one-stepcondensation of the corresponding aldehyde and hydrazine. Moreover, byintroduction of a third aromatic ring system into the shorter inhibitorsit is possible to construct compounds that fit all three pockets of thetarget site of the enzyme (the S2, S1 and S1' subsites).

On the basis of these structure/activity studies, a broad class ofmetazoan parasite protease inhibitors have been identified as ofparticular utility in accordance with the present invention having thegeneral structure

    A-X-B

wherein A is a substituted or unsubstituted homoaromatic orheteroaromatic ring system comprising one to three rings which binds tothe S2 subsite;

B is a substituted or unsubstituted homoaromatic or heteroaromatic ringsystem comprising one to three rings which binds to the S1 or S1'subsite; and

X is a linker comprising a substantially planar linear array with abackbone of four to eight atoms in length.

In a first preferred class of inhibitors in accordance with the presentinvention, B binds to the S1' subsite and X is a linker with a backboneof six to eight atoms in length. In a second preferred class ofinhibitors in accordance with the present invention, B binds to the S1subsite and X is a linker with a backbone of about 4 atoms in length. Ina preferred subclass of this second preferred class, B has thestructure--B'-X'-B", wherein B' is an aromatic ring system which bindsto the S1 subsite, B" is an aromatic ring system which binds to the S1'subsite, and X' is a direct bond or a linker with a backbone of one tothree atoms in length.

The ring system A is preferably a one- or two-ring homoaromatic (e.g.,phenyl, 1-naphthyl, 2-naphthyl, etc.) or heteroaromatic group which hasan affinity for the S2 subsite of the metazoan parasite protease. Thering system A may be unsubstituted; preferably, however, the ring systemA bears at least one non-interfering substituent (as hereinafterdefined) which does not interfere with, and may actually promote, thebinding in the S2 subsite via interactions with the side chains withhydrophobic features, side chains and polypeptide backbone elements withdonor and acceptor sites for hydrogen bonds and side chains with formalcharges for electrostatic interactions characteristic of the S2 subsite.A particularly preferred class of ring systems A are those that aresubstituted by one or more hydroxyl groups, as hydroxyl is believed tobe especially effective in promoting binding to the subsite; groups withup to three hydroxyl substituents have shown good affinity for the S2subsite.

Particular examples of suitable A systems include, but are not limitedto, the following: phenyl; 1-naphthyl; 1-isoquinolyl; 1-phthalazinyl;3-coumarinyl; 9-phenanthryl; and 1-quinolyl. Again, all of these ringsystems may be unsubstituted or substituted by one or morenon-interfering substituents as hereinafter defined. Among the phenylsystems, particular embodiments of interest include, but are not limitedto, the following: 1-hydroxyphenyl; 2,3-dihydroxyphenyl;1,2,3-trihydroxyphenyl; and 3-di-(lower-alkyl)-aminophenyl. Particularlypreferred as ring system A is 1-naphthyl: ##STR1## In addition to theunsubstituted 1-naphthyl group, the following substituted 1-naphthylsystems have been found especially useful in inhibitors within the scopeof the present invention: R¹ =OH; R³ =OH; R¹ =lower alkoxy (e.g.,--OCH₃); R³ =lower alkoxy (e.g., --OCH₃); R³ =lower alkyl (e.g., --CH₃);R³ =di-(lower-alkyl)-amino (e.g., --N(CH₃)₂ ; R⁴ =NO₂ ; R⁷ =COOH; R¹, R³=OH; R¹, R² =OH; R², R⁷ =OH; R¹, R⁵ =OH; R³, R⁶ =OH; R¹, R⁷ =OH; R³, R⁵=OH; R¹, R⁴ =OH; R¹, R⁶ =OH; R¹ =OH, R⁵ =NO₂ ; and R¹ =OH, R⁵ =Br.

The choice of a particular structure for use as ring system B depends tosome extent on whether the inhibitor is being designed for use to blockthe S1' subsite (i.e., an inhibitor comprising a longer linker) or theS1 subsite (i.e., an inhibitor comprising a shorter linker). In general,it has been determined that for binding to the S1' subsite, a two-ringsystem (for example, as described for use as Group A) is more effectivethan a one-ring system. In addition, ring systems bearing a substituentcontaining a heteroatom (O, N) and/or heterocyclic systems with chargedatoms (in particular, quinoline) are preferred. On the other hand, theone- and two-ring systems are essentially equally effective for use inbinding to the S1 subsite.

For use in binding to the S1 subsite, the following multiple-ringsystems are preferred, each of which again may be unsubstituted orsubstituted by one or more non-interfering substituents (as hereinafterdefined): 1-naphthyl; 2-naphthyl; 2-quinolyl; 3-quinolyl; 6-coumarinyl;2-chromonyl (4-oxo-1,4-chromen-2-yl). Particularly preferred are anumber of substituted 2-naphthyl systems, including but not limited tothe following: 3-hydroxy-2-naphthyl; 3-(lower-alkoxy)-2-naphthyl;5-hydroxy-2-naphthyl; and 4,7-dibromo-2-naphthyl. In addition, phenyland substituted phenyl groups are also useful in binding to the S1subsite. The multiple-ring systems (and in particular, two-ring systems)previously described as useful in binding to the S2 and S1 subsites arealso presently preferred for binding to the S1' subsite.

The choice of linker X, as previously indicated, is dependent upon theparticular subsites to which the particular inhibitor is targeted. Forinhibitors directed to the S2 and S1' subsites, linkers containing abackbone consisting of six-eight atoms are particularly preferred; forinhibitors directed to the S2 and S1 subsites, however, linkerscontaining a backbone consisting of four atoms are especially useful. Inall cases, the backbone may comprise one or more heteroatoms (N, O, S,etc.) in addition to carbon. Moreover, particularly suitable linkershave a relatively planar character induced by multiple bonds either inthe backbone (e.g., --C═C═, --C═N--, etc.) or external thereto (e.g.,--C(═O)--, --C(═S)--, etc.). Most preferred are linkers wherein there isan extended, substantially conjugated system of multiple bonds andheteroatoms with unshared electron pairs (e.g., O, N, S, etc.) whichtends to maintain a relatively planar arrangement. However, linkers inwhich the backbone includes an alicyclic group (e.g., 1.3- or1,4-cyclohexyl) are also contemplated as within the scope of the presentinvention. These linkers may also be substituted with one or morenon-interfering groups (as hereinafter defined).

Examples of suitable linkers wherein the backbone consists of four atomsinclude the following (the atoms constituting the linker backbone beingindicated in bold face): ##STR2## wherein R is hydrogen or lower alkyl;##STR3## wherein R is hydrogen or lower alkyl; ##STR4## wherein R ishydrogen or lower alkyl, R' is hydrogen or lower alkyl, and Y is O or S;and ##STR5## wherein R is hydrogen or lower alkyl, R' is hydrogen orlower alkyl, and Y is O or S. The first of the listed structures isparticularly preferred, in view of the ease of synthesis of compoundscontaining this structure by reaction of the appropriate aldehyde andhydrazide.

For use in inhibitors targeted to the S2 and S1' subsites, linkerswherein the backbone contains 6 to 8 atoms are particularly suitable.Structures of interest include the following: ##STR6## wherein R ishydrogen or lower alkyl and R' is hydrogen or lower alkyl; ##STR7##wherein R, R' and R" are independently selected from hydrogen and loweralkyl; ##STR8## wherein the cyclohexyl group is unsubstituted orsubstituted by one or more non-interfering substituents (as hereinafterdefined); and ##STR9## wherein R and R' are independently selected fromhydrogen and lower alkyl. Of course, other structures containingbackbones having the requisite number of atoms and exhibitingsubstantial planar character would be immediately apparent to thoseskilled in the art and are contemplated as suitable for use inaccordance with the present invention.

As previously noted, both the ring systems A and B and the linker X maysuitably bear one or more non-interfering substituents. For purposes ofthe present invention, a non-interfering substituent is defined as onewhich does not interfere with bonding of the ring structures to theactive site of the enzyme due to steric and/or electronic factors; insome cases, the presence of particular non-interfering substituents isbelieved to promote bonding by interaction of these substituents withstructural elements of the enzyme in the proximity of the active site.In most instances, the primary consideration with respect to possiblesubstituents is a steric one; for the most part, relatively bulkysubstituents are not particularly preferred for use in the inhibitors ofthe present invention. Suitable non-interfering substituents include,but are not limited to, the following: hydroxyl, including protectedhydroxyl (i.e., a hydroxyl group which is protected by a suitableart-recognized protective group); lower alkyl; lower alkoxy; amino,mono- and di-(lower-alkyl)-amino; --COOH and --COOR', wherein R' islower alkyl or aryl; --NO₂ ; halogen (in particular, Cl, F and Br); aryl(in particular, phenyl and benzyl); and aryloxy (in particular, phenoxyand benzyloxy). For purposes of the present invention, by lower alkyl ismeant an alkyl group of one to five, and preferably one to three, carbonatoms.

Particular inhibitors suitable for use in the compositions and methodsof the present invention include a number of general classes ofcompounds which have been investigated in some detail. One such class ofcompounds wherein X has a backbone of four atoms has the generalformula: ##STR10## wherein the substitution pattern is advantageouslyselected from those reported in Table 1.

                  TABLE 1                                                         ______________________________________                                        COMPOUND     SUBSTITUENTS                                                     ______________________________________                                        I193A        2-OH, 3'-OH                                                      III23A       2-OH, 6-NO.sub.2, 3'-OH                                          III41A       2,3-OH, 3'-OH                                                    III43A       2,4-OH, 3'-OH                                                    III45A       2,7-OH, 3'-OH                                                    III53A       2-OMe, 3'-OMe, NMe                                               III55A       2-OMe, 3'-OH                                                     III59A       4,8-OH, 3'-OH                                                    III71A       4-F, 3'-OH                                                       III79A       2,6-OH, 3'-OH                                                    III81A       4,7-OH, 3'-OH                                                    III91A       5-NO.sub.2, 3'-OH                                                III93A       2-H, 3'-OH                                                       III95A       4-Me, 3'-OH                                                      III97A       4-OH, 3'-OH                                                      III115A      4-NMe.sub.2, 3'-OH                                               III128A      2-OH, 3'-H                                                       III130A      2-OMe, 2'-OMe                                                    III127A      2-OH, 1'-OH                                                      III132A      8-COOH, 3'-OH                                                    III133A      2-OH, 3'-OH, 7'-OMe                                              III134A      2-OH, 3',5'-OH                                                   III135A      2-OH, 1',4'-OH, OMe                                              III138A      4-OMe, 3'-OH                                                     III144A      2,8-OH, 3'-OH                                                    III145A      4,6-OH, 3'-OH                                                    III146A      2,5-OH, 3'-OH                                                    III151A      2-OH, 6'-OH                                                      III152A      2-OH, 1'-Me                                                      III153A      2-OH, 8'-Ph                                                      III154A      2-OH, 3'-Br                                                      III155A      2-OH, 3'-NHMe                                                    IV17A        2-OH, 3'-OH, 4'-N.sub.2 (2-Cl, 5-CF.sub.3 -Phe)                  ______________________________________                                    

Another class of compounds wherein X also has a backbone of four atomshaving particular utility as inhibitors of metazoan parasite proteaseshave the general formula ##STR11## wherein the substitution pattern isadvantageously selected from those reported in Table 2.

                  TABLE 2                                                         ______________________________________                                        Compound          Substituents                                                ______________________________________                                        III85A            2-OH, 4'-Ph                                                 III87A            2-OH, 3'-OPh                                                III103A           2-OH, 2'-OPh                                                III109A           2-OH, 2'-OH                                                 IV36A             2,4-OH, 2'-OH                                               IV37A             2,7-OH, 2'-OH                                               IV38A             2,6-OH, 2'-OH                                               IV39A             2-OH, 6-NO.sub.2, 2'-OH                                     IV44A             2-OH, 2',4'-OH                                              IV45A             2-OH, 3',4'-OH                                              IV46A             2-OH, 2'-H                                                  IV47A             2-OH, 3',5'-OH                                              IV49A             2-OH, 2'-OMe                                                IV50A             2-OH, 2'-F                                                  IV51A             2-OH, 3',4'-NH.sub.2                                        ______________________________________                                    

Yet another class of compounds wherein X has a backbone of four atomshaving particular utility as inhibitors for use in accordance with thepresent invention have the general formula ##STR12## wherein thesubstitution pattern is advantageously as indicated in Table 3.

                  TABLE 3                                                         ______________________________________                                        Compound     Substituents                                                     ______________________________________                                        III107A      2-OH, 3'-OH                                                      III111A      4-NBu.sub.2, 3-OH                                                III113A      4-(3-dimethylpropoxy), 3'-OH                                     IV7A         3-OMe, 4-(4-NO.sub.2 -benzyloxy), 3'-OH                          IV34A        3-OH, 4-NO.sub.2, 3'-OH                                          IV35A        2,3,4-OH, 3'-OH                                                  ______________________________________                                    

The following additional examples of inhibitors wherein X has a backboneof four atoms are illustrative of the range of structures which may beemployed as ring system A, ring system B and linker X. Of course,analogous ring systems and substitution patterns other than thosedepicted herein would be immediately recognized by those working in thefield as equivalent to the structures illustrated and are thus are alsocontemplated as within the scope of the present invention. ##STR13##

The following structures are illustrative of inhibitors wherein X has abackbone of 6-8 carbon atoms. Once again, these examples should beviewed as merely illustrative of the range of structures which may beemployed as ring system A, ring system B and linker X. Analogous ringsystems and substitution patterns other than those depicted herein arecontemplated as within the scope of the present invention. ##STR14##

Finally, the following structures illustrate inhibitors of the generalformula

    A-X-B'-X'-B"

designed to bond to the S2, S1 and S1' subsites. In these structures, X'is exemplified as follows: a direct ring-to-ring bond; a single-atombackbone linker (e.g., --CH₂ --); and a two-atom backbone linker (e.g.,--CH₂ --O--and--N=N--). Other X' linkers as hereinbefore specified wouldbe readily apparent substitutes for these exemplary structures. Asindicated with respect to the previous structures, these examples shouldbe viewed as merely illustrative of the range of structures which may beemployed as ring system A, ring system B and linker X, and once againanalogous ring systems and substitution patterns other than thosedepicted herein are contemplated as within the scope of the presentinvention. ##STR15##

Many of the inhibitors employed in accordance with the present inventionare either known compounds (some of which are commercially available) ormay be readily prepared in a manner known per se from heretofore knownand/or commercially-available compounds. The following general schemesillustrate some particularly advantageous synthetic routes; alternativesyntheses will of course be readily apparent to those skilled in thefield of synthetic organic chemistry.

The synthesis of symmetrical bis-hydrazides and bis-hydrazones withinthe scope of the present invention (i.e., compounds in which ring systemA and ring system B are identical) may readily be effected as follows:##STR16## In these illustrations, both ring systems are denominated "A"for clarity. The corresponding aldehydes are either commerciallyavailable or may be readily prepared from commercially-availablematerials by an appropriate reaction scheme (e.g., treatment of theappropriately-substituted precursor with n-butyl lithium and DMF).

The following scheme illustrates a preferred method for synthesis ofasymmetrical bis-hydrazides. Although the B ring system is introducedfirst according to the scheme as illustrated it is of course apparentthat the A ring system could equally well be introduced first. ##STR17##

The following scheme illustrates a preferred method for synthesis ofasymmetrical bis-hydrazones. Once again, the B ring system is introducedfirst according to the scheme as illustrated, but it is of courseapparent that the A ring system could be the first introduced. ##STR18##

As previously noted, several classes of inhibitors wherein the linker Xhas a backbone of four atoms in length are of particular interestbecause they can be synthesized in a one-step condensation of analdehyde and a hydrazine or hydrazine. The following schemes illustratesuch condensations: ##STR19##

The following two schemes illustrate the synthesis of inhibitorscomprising particular heterocyclic ring A systems: ##STR20## By analogy,a variety of different heterocyclic systems having a desiredsubstitution pattern may be prepared for reaction with the appropriatehydrazide.

Finally, the following two schemes illustrate a preferred approach forsynthesis of inhibitors comprising ring systems A, B' and B": ##STR21##Once again, it would be readily apparent to those skilled in the artthat by choice of suitable precursors, other ring systems A, B' and B"may be joined in a similar manner.

The inhibitors employed in the compositions and methods of the presentinvention are typically administered in conjunction with a suitablecarrier or adjuvant. It is presently preferred that the inhibitors beadministered in an aqueous solution (e.g., buffered saline); however,other suitable excipients and adjuvants would be readily apparent tothose of skill in the art. The compositions of the invention may beadministered by a wide variety of known routes of administration (e.g.,orally, intravenously, subcutaneously, etc.). The inhibitors aresuitably administered at a dosage of about 0.01 to about 10 μM, andpreferably about 0.01 to about 1 μM, per kilogram of body weight of thepatient per day. Of course, as would be appreciated by those of skill inthe art, the optimum dosage for treatment of any given parasiticinfection with a composition of the present invention comprising one ormore specific inhibitors as described herein may readily be determinedempirically.

The inhibitors determined to be effective in accordance with the presentinvention exhibit a surprising specificity for the malarial protease andother evolutionarily-related metazoan parasite proteases. These metazoanparasite proteases are distinct from proteases found in the parasitichosts (i.e., mammals), particularly with respect to the chemicalenvironments of the active sites of the respective enzymes. In view ofthe significant differences between corresponding subsites in themammalian and the metazoan parasite proteases, the inhibitors of thepresent invention do not in general inhibit the activity of the host'sessential proteases.

For example, the malarial enzyme has an asparagine at position 133, akey residue for determining the specificity of bonding at the S2subsite; for most other non-parasitic cysteine proteases, however, thisresidue tends to be either branched hydrophobic or alanine. Specificinteractions of the inhibitors in accordance with the present inventionwith the asparagine increase both specificity and potency. Anothermodulating residue is glutamic acid at position 205 in the malarialenzyme. The side chain rotamer located at the base of the S2 bindingpocket is postulated to change depending upon the nature of theinteraction at S2. If the substituent is hydrophobic, the glutamic acidpoints away from the S2 pocket and presumably interacts with solvent;however, when the substituent is basic, the glutamic acid is thought topoint towards the S2 pocket and provide a crucial interaction with thepositive charge. Inhibitors that exploit this interaction (i.e., thosewherein ring system A has some basic characteristics as a part of thering system and/or by virtue of the substitution pattern) are thus ofparticular interest.

The invention may be better understood with reference to theaccompanying examples, which are intended for purposes of illustrationonly and should not be construed as in any sense limiting the scope ofthe present invention as defined in the claims appended hereto.

EXAMPLES

In the following examples, melting points were determined on a Thomascapillary melting point apparatus and are uncorrected. Proton and carbonNMR spectra were obtained at 300 and 75 MHz, respectively, in DMSO-d⁶ ona GE QE-300 instrument. Mass spectra (MS) were recorded on a VG-70 massspectrometer equipped with a Hewlett-Packard 5890A GC. All aldehydes andhydrazides were obtained commercially except for thedihydroxynaphthaldehydes, which were prepared from the correspondingcommercially-available dihydroxynaphthalenes according to the literatureprocedure [Morgan, G. T. & Vining, D. C., J. Chem. Soc 119, 177 (1921)].2-Hydroxy-1-naphthaldehyde was recrystallized from EtOH/H₂ O (8:2, v/v).

Example 1

Synthesis of 2-hydroxy-1-naphthaldehyde azine (I73A)

In the general procedure for condensation of aldehyde with hydrazine, toa solution of the aldehyde (1 mmol) in methanol (20 mL) was added thecorresponding hydrazine or acyl hydrazine (1 mmol) in one portion. Theresulting mixture was heated to 65° C. for 3 hours. In most cases, aprecipitate was observed after 10 minutes. The precipitate was filtered,washed with hot methanol (50 mL), and dried in vacuum (2 mmHg). Ifneeded, additional purification was performed by recrystallization usingappropriate solvents. Following this general procedure, condensation of2-hydroxy-1-naphthaldehyde and hydrazine gave 2-hydroxy-1-naphthaldehydeazine as a yellow solid (98%): mp>300° C. (dec); ¹ H NMR d 12.90 (br s,2 H), 9.99 (s, 2 H), 8.64 (d, 2 H, J=8.7 Hz), 8.03 (d, 2 H, J=9.0 Hz),7.92 (d, 2 H, J=7.8 Hz), 7.61 (t, 2 H, J=7.6 Hz), 7.44 (t, 2 H, J=7.4Hz), 7.28 (d, 2 H, J =8.7 Hz); HRMS Calcd for C₂₂ H₁₆ N₂ O₂ :340.1212,found 340.1195; Anal. (C₂₂ H₁₆ N₂ O₂) C, H, N.

Example 2

Synthesis of Oxalic bis(2-hydroxy-l-phenylmethylene) hydrazide (I75A)

Following the general procedure of Example 1, condensation of salicylicaldehyde and oxalic dihydrazide gave a white solid (97%): mp>300° C.; ¹H NMR d 12.65 (s, 2 H), 11.00 (s, 2 H), 8.79 (s, 2H), 7.54 (d, 2 H,J=7.5 Hz), 7.32 (t, 2 H, J=7.6 Hz), 6.91 (m, 4 H); ¹³ C NMR d 157.58,155.83, 151.07, 131.99, 129.36, 119.44. 118.59, 116.45; HRMS Calcd forC₁₆ H₁₄ N₄ O₄ :326.1015, found 326.0997; Anal. (C₁₆ H₁₄ N₄ O₄) C, H, N.

Example 3

Synthesis of oxalic bis(9-phenanthrylmethylene) hydrazide (I77A)

Following the general procedure of Example 1, condensation ofphenanthrene-9-carboxaldehyde and oxalic dihydrazide gave a white solid(97%): mp>300° C.; MS (CI) m/e 495.4 (M+H)+; Anal. (C₃₂ H₂₂ N₄ O₂) C, H,N.

Example 4

Synthesis of 3-hydroxy-2-naphthoic (2-hydroxy-1-naphthylmethylene)hydrazide (I93A)

Following the general procedure of Example 1, condensation of2-Hydroxy-1-naphthaldehyde and 3-hydroxy-2-naphthoic hydrazide gave awhite solid (97%): mp >300° C.; ¹ H NMR d 12.70 (s, 1 H), 12.25 (br s, 1H), 11.37 (br s, 1 H); 9.58 (s, 1 H), 8.52 (s, 1 H), 8.33 (d, 1 H, J=8.7Hz), 7.94 (d, 2 H, J=8.7 Hz), 7.89 (d, 1 H, J=8.1 Hz), 7.78 (d, 1 H,J=8.4 Hz), 7.58 (t, 1 H, J=7.6 Hz), 7.52 (t, 1 H, J=7.5 Hz), 7.41 (m, 2H), 7.37 (s, 1 H), 7.25 (d, 1 H, J=9.0 Hz); ¹³ C NMR d 163.05, 158.12,153.88, 147.57, 135.94, 132.90, 131.68, 130.62, 128.90, 128.68, 128.33,127.79, 127.73, 126.81, 125.85, 123.86, 123.54, 120.98, 119.84, 118.87,110.62, 108.61; HRMS Calcd for C₂₂ H₁₆ N₂ O₃ :356.1161, found 356.1151;Anal. (C₂₂ H₁₆ N₂ O₃) C, H, N.

Example 5

Synthesis of Bis(2-hydroxy-1-naphthylmethylene) carbohydrazide (197A)

Following the general procedure of Example 1, condensation of2-hydroxy-1-naphthaldehyde and carbohydrazide gave a white solid (97%):mp>285° C. (dec); ¹ H NMR d 11.90 (br s, 2 H); 11.05 (s, 2 H), 9.23 (s,2 H), 8.33 (d, 2 H, J=8.4 Hz), 7.88 (d, 2 H, J=9.0 Hz), 7.87 (d, 2 H,J=7.8 Hz), 7.59 (t, 2 H, J=7.6 Hz), 7.38 (t, 2 H, J=7.5 Hz), 7.22 (d, 2H, J=9.0 Hz); ¹³ C NMR d 156.71, 151.56, 143.24, 131.88, 131.50, 128.72,127.87, 127.56, 123.35, 121.58, 118.61, 109.40; MS (CI) m/e 399.0 (M+H)+; Anal. (C₂₃ H₁₈ N₄ O₃ 0.3 H₂ O) C, H, N.

Example. 6

Synthesis of Oxalic bis(3-indolylmethylene) hydrazide (I135A)

Following the general procedure of Example 1, condensation ofindole-3-carboxaldehyde and oxalic dihydrazide gave a white solid (97%):mp>285° C. (dec); ¹ H NMR d 11.93 (br s, 2 H), 11.66 (s, 2 H), 8.77 (s,2 H), 8.28 (d, 2 H, J=7.5 Hz), 7.84 (d, 2 H, J=2.1 Hz), 7.46 (d, 2 H,J=7.8 Hz), 7.16 (m, 4 H); ¹³ C NMR d 156.01, 147.85, 137.09, 131.13,124.37, 122.77, 121.93, 120.63, 111.95, 111.48; HRMS Calcd for C₂₀ H₁₆N₆ O₂ :372.1341, found 372.1335; Anal. (C₂₀ H₁₆ N₆ O₂ 0.3 H₂ O) C, H, N.

Example 7

Synthesis of 3-Hydroxy-2-naphthoic (2,3-dihydroxy-1-naphthylmethylene)hydrazide (III41A)

Following the general procedure of Example 1, condensation of 2,3-dihydroxy-1-naphthaldehyde and 3-hydroxy-2-naphthoic hydrazide yieldeda yellow crystal (18%) [recrystallized from dioxane/H₂ O (1:1, v/v)]:mp>285° C.; ¹ H NMR d 13.16 (br s, 1 H), 12.27 (br s, 1 H), 11.30 (br s,1 H), 9.74 (br s, 1 H), 9.57 (s, 1 H), 8.53 (s, 1 H), 8.17 (d, 1 H,J=8.2 Hz), 7.95 (d, 1 H, J=8.2 Hz), 7.79 (d, 1 H, J=8.0 Hz), 7.70 (d, 1H, J=7.7 Hz), 7.54 (t, 1 H, J=7.4 Hz), 7.42-7.25 (m, 5 H); HRMS Calcdfor C₂₂ H₁₆ N₂ O₄ :372.1110, found 372.1103; Anal. (C₂₂ H₁₆ N₂ O₄ H₂ O)C, H, N.

Example 8

Synthesis of 3-Hydroxy-2-naphthoic (2.4-dihydroxy-1-naphthylmethylene)hydrazide (III43A)

Following the general procedure of Example 1, condensation of 2,4-dihydroxy-1-naphthaldehyde and 3-hydroxy-2-naphthoic hydrazide yieldeda yellow crystal (83%) [recrystallized from DMSO/H₂ O (8:2, v/v)]:mp>280° C. (dec); ¹ H NMR d 12.42 (br s, 1 H), 11.65 (s, 1 H), 10.96 (brs, 1 H), 10.59 (s, 1 H), 8.99 (s, 1H), 8.07 (s, 1H), 7.77 (d, 1 H, J=8.4Hz), 7.67 (d, 1 H, J=8.4 Hz), 7.48 (d, 1 H, J=8.1 Hz), 7.32 (d, 1H,J=8.4 Hz), 7.13 (t, 1H, J=7.5 Hz), 7.06 (t, 1 H, J=7.5 Hz), 6.91 (s, 1H), 6.90 (m, 2 H); ¹³ C NMR d 162.92, 160.47. 157.70, 154.20, 148.22,135.97, 132.99, 130.33, 128.72, 128.33 (2 C), 126.84, 125.92, 123.91,122.94, 122.55, 120.80, 120.32, 119.66, 110.70, 101.38, 100.49; HRMSCalcd for C₂₂ H₁₆ N₂ O₄ :372.1110, found 372.1095; Anal. (C₂₂ H₁₆ N₂ O₄2DMSO H₂ O) C, H, N.

Example 9

Synthesis of 3-Hydroxy-2-naphthoic (2, 7-dihydroxy-1-naphthylmethylene)hydrazide (III45A)

Following the general procedure of Example 1, condensation of 2,7-dihydroxy-1-naphthaldehyde and 3-hydroxy-2-naphthoic hydrazide yieldedyellow crystals (83%) [recrystallized from DMSO/H₂ O (8:2, v/v)]: mp225°C. (dec); ¹ H NMR d 12.75 (s, 1 H), 12.24 (s, 1 H), 11.27 (s, 1 H), 9.93(s, 1 H), 9.40 (s, 1H), 8.50 (s, 1 H), 7.95 (d, 1 H, J=8.1 Hz), 7.80 (d,2 H, J=8.7 Hz), 7.73 (d, 1 H, J=8.7 Hz), 7.54 (t, 1 H, J=7.5 Hz), 7.39(t, 1 H, J=7.5 Hz), 7.37 (s, 1 H), 6.98 (d, 2 H, J=8.7 Hz); HRMS Calcdfor C₂₂ H₁₆ N₂ O₄ :372.1110, found 372.1099; Anal. (C₂₂ H₁₆ N₂ O₄ 1.4DMSO) C, H, N.

Example 10

Synthesis of 3-Hydroxy-2-naphthoic (2, 6-dihydroxy-1-naphthylmethylene)hydrazide (III79A)

Following the general procedure of Example 1, condensation of 2,6-dihydroxy-1-naphthaldehyde and 3-hydroxy-2-naphthoic hydrazide yieldeda yellow solid (72%): mp>280° C.; ¹ H NMR d 11.89 (s, 1 H), 11.75 (br s,1 H), 10.90 (br s, 1 H), 9.14 (s, 1 H), 9.03 (s, 1 H), 8.06 (s, 1 H),7.75 (d, 1 H, J=9.0 Hz), 7.48 (d, 1 H, J=8.4 Hz), 7.33 (d, 1 H, J=8.4Hz), 7.28 (d, 1 H, J=9.0 Hz), 7.07 (t, 1 H, J=7.6 Hz), 6.92 (t, 1 H,J=7.4 Hz), 6.75-6.67 (m, 3 H), 6.90 (m, 2 H); ¹³ C NMR d 163.06, 155.90,153.93, 153.44, 147.82, 135.94, 131.34, 130.57, 129.35, 128.69, 128.34,126.81, 125.87, 125.66, 123.88, 122.58, 119.86, 119.68, 119.04, 110.62,110.45, 108.82; HRMS Calcd for C₂₂ H₁₆ N₂ O₄ :372.1110, found 372.1095;Anal. (C₂₂ H₁₆ N₂ O₄ 0.4 H₂ O) C, H, N.

Example 11

Synthesis of 3-Hydroxy-2-naphthoic (1-naphthylmethylene) hydrazide(III93A)

Following the general procedure of Example 1, condensation of1naphthaldehyde and 3-hydroxy-2-naphthoic hydrazide gave a pale yellowsolid (76%): mp 214° C.; ¹ H NMR d 11.73 (br s, 1 H), 11.05 (br s, 1 H),8.72 (s, 1 H), 8.52 (d, 1 H, J=8.4 Hz), 8.13 (s, 1 H), 7.60-6.80 (m, 11H); ¹³ C NMR d 164.00, 154.31, 148.56, 135.99, 133.57, 130.80, 130.40.130.30, 129.48, 128.81, 128.78, 128.29, 128.12, 127.37, 126.85, 126.31.125.89, 125.54, 124.39, 123.84, 120.13, 110.75; HRMS Calcd for C₂₂ H₁₆N₂ O₂ :340.1212, found 340.1208; Anal. (C₂₂ H₁₆ N₂ O₂) C, H, N.

Example 12

Synthesis of 3-Hydroxy-2-naphthoic (2-Hydroxy-1phenylmethylene)hydrazide (III107A)

Following the general procedure of Example 1, condensation of salicylicaldehyde and 3-hydroxy-2-naphthoic hydrazide gave a pale yellow solid(94%): mp >285° C.; ¹ H NMR d 11.69 (br s, 1H), 10.97 (br s, 1 H), 10.79(s, 1 H), 8.24 (s, 1 H), 8.02 (s, 1 H), 7.45 (d, 1 H, J=8.1 Hz), 7.31(d, 1 H, J=8.1 Hz), 7.12 (dd, 1 H, J=7.8, 0.9 Hz), 7.05 (t, 1 H, J=7.5Hz), 6.89 (s, 1 H), 6.86 (m, 2H), 6.51 (d, 1 H, J=8.1 Hz), 6.48 (t, 1H,J=7.5 Hz); ¹³ C NMR d 163.62, 157.54, 154.08, 148.83, 135.94, 131.64,130.37, 129.49, 128.70, 128.33, 126.79, 125.89, 123.86, 119.99, 119.44,118.68, 116.48, 110.63; HRMS Calcd for C₁₈ H₁₄ N₂ O₃ :306.1004, found306.0977; Anal. (C₁₈ H₁₄ N₂ O₃) C, H, N.

Example 13

Synthesis of Salicylic (2-hydroxy-1-naphthylmethylene) hydrazide(III109A)

Following the general procedure of Example 1, condensation of2-hydroxy-1-naphthaldehyde and salicylic hydrazide yielded a pale yellowsolid (74%): mp 265° C.; ¹ H NMR d 12.32 (s, 1 H), 11.65 (br s, 1 H),11.36 (br s, 1 H), 9.09 (s, 1 H), 7.84 (d, 1 H, J=7.5 Hz), 7.43 (m, 3H), 7.13 (t, 1 H, J=6.0 Hz), 7.01 (t, 1 H, J=6.6 Hz), 6.83 (t, 1H, J=6.9Hz), 6.77 (d, 1H, J=8.4 Hz), 6.57 (m, 2 H); ¹³ C NMR d 163.72, 158.58,157.88, 147.40, 133.75, 132.63, 131.42, 128.64, 128.49, 127.53, 127.44,123.27, 120.65, 118.87, 118.61, 117.04, 115.35, 108.32; HRMS Calcd forC₁₈ H₁₄ N₂ O₃ :306.1004, found 306.0985; Anal. (C₁₈ H₁₄ N₂ O₃) C, H, N.

    ______________________________________                                        Analytical Data                                                                             elemental analysis                                              entry                C %     H %     N %                                      ______________________________________                                        I73A            calcd    77.63   4.74  8.23                                   C.sub.22 H.sub.16 N.sub.2 O.sub.2                                                             found    77.71   4.90  7.93                                   I75A            calcd    58.89   4.32  17.17                                  C.sub.16 H.sub.14 N.sub.4 O.sub.4                                                             found    58.79   4.59  16.93                                  I77A            calcd    77.16   4.53  11.25                                  C.sub.32 H.sub.22 N.sub.4 O.sub.2.0.3H.sub.2 O                                                found    76.94   4.13  11.53                                  I93A            calcd    74.15   4.53  7.86                                   C.sub.22 H.sub.16 N.sub.2 O.sub.3                                                             found    73.80   4.73  7.78                                   I97A            calcd    68.41   4.64  13.74                                  C.sub.20 H.sub.16 N.sub.6 O.sub.2.0.3H.sub.2 O                                                found    68.33   4.59  13.97                                  I135A           calcd    63.58   4.43  22.24                                  C.sub.22 H.sub.16 N.sub.2 O.sub.4.H.sub.2 O                                                   found    63.58   4.48  22.13                                  III41A          calcd    67.69   4.65  7.18                                   C.sub.22 H.sub.16 N.sub.2 O.sub.4.2DMSO.H.sub.2 O                                             found    68.09   4.35  7.13                                   III43A          calcd    57.13   5.53  5.12                                   C.sub.22 H.sub.16 N.sub.2 O.sub.4.4DMSO                                                       found    57.45   5.33  5.11                                   III45A          calcd    61.83   5.10  5.81                                   C.sub.22 H.sub.16 N.sub.2 O.sub.4.0.4H.sub.2 O                                                found    61.65   4.69  5.82                                   III79A          calcd    69.61   4.46  7.38                                   C.sub.22 H.sub.16 N.sub.2 O.sub.4                                                             found    69.64   4.81  7.33                                   III93A          calcd    77.63   4.74  8.23                                   C.sub.18 H.sub.14 N.sub.2 O.sub.3                                                             found    77.29   4.88  8.27                                   III107A         calcd    70.58   4.61  9.15                                   C.sub.18 H.sub.14 N.sub.2 O.sub.3                                                             found    70.27   4.70  9.06                                   III109A         calcd    70.58   4.61  9.15                                   C.sub.16 H.sub.14 N.sub.2 O.sub.3                                                             found    70.59   4.69  9.04                                   ______________________________________                                    

Example 14

Trophozoite cysteine protease inhibitor studies

Enzyme activity was measured with the fluorogenic substrateZ-Phe-Arg-AMC as described in the literature [Rosenthal, P. J. et al.,Mol. Biochem. Parasitol. 35, 177 (1989)]. Trophozoite extracts wereincubated with reaction buffer (in 0.1 M sodium acetate, 10 mMdithiothreitol, pH 5.5) and an appropriate concentration of inhibitorfor 30 minutes at room temperature. Z-Phe-Arg-AMC (50 μM finalconcentration) was then added and fluorescence (380 nM excitation, 460nM absorbance) was measured continuously over 30 seconds. The slope offluorescence over time for each inhibitor concentration was comparedwith that of controls in multiple assays, and the IC₅₀ was determinedfrom plots of percent control activity over inhibitor concentration. Theresults are illustrated in FIG. 1, wherein the points are the mean of 8assays and the error bars are the standard deviation of the sample. TheIC₅₀ values for inhibitors in accordance with the present invention arereported in Table 4.

                  TABLE 4                                                         ______________________________________                                        COMPOUND        IC50 (μm)                                                  ______________________________________                                        I38A            60                                                            I40A            18                                                            I42A            >60                                                           I46A            33                                                            I56A            50                                                            I73A            10                                                            I75A            >60                                                           I77A            10                                                            I83A            16                                                            I89A            40                                                            I93A            10                                                            I97A            60                                                            I99A            60                                                            I101A           >60                                                           I115A           12                                                            III111A         20                                                            III113A         40                                                            III115A         8                                                             III117A         15                                                            III127A         20                                                            III128A         17                                                            III129A         20                                                            III130A         14                                                            III132A         17                                                            III133A         9                                                             III134A         20                                                            III135A         23                                                            III136A         11                                                            III138A         15                                                            III139A         15                                                            ______________________________________                                    

Example 15

Effect of oxalic his (2-hydroxy-1-naphthylmethylene)hydrazide) on ³H-hypoxanthine uptake as a measure parasite metabolism

³ H-hypoxanthine uptake was measured based on a modification of themethod of Desjardins et al. [Antimicrob. Agents Chemother. 16, 710-718(1979)]. Microwell cultures of synchronized ring-stage P. falciparumparasites were incubated with inhibitor in DMSO (10% finalconcentration) for 4 hours. ³ H-hypoxanthine was added (1μCi/microwellculture) and the cultures were maintained for an additional 36 hours.The cells were then harvested and deposited onto glass fiber filterswhich were washed and dried with ethanol. ³ H-hypoxanthine uptake wasquantitated by scintillation counting. The uptake at each inhibitorconcentration was compared with that of controls and the IC₅₀ wasdetermined from plots of percent control uptake over inhibitorconcentration. The results are illustrated in FIG. 2.

Example 16

Effect of oxalic bis [(2-hydroxy-1-naphthylmethylene)hydrazide]onability of parasites to invade red blood cells

The FCR3 strain of Plasmodium falciparum was maintained in Type O+humanerythrocytes (American Red Cross) at 4% hematocrit in RPMI 1640 medium(Gibso supplemented with 10% human serum, hypoxanthine, and gentamicin).Asynchronous infected cells were centrifuged at 500 g for 5 minutes andthe supernatant aspirated. Sorbitol (5% in dH20) was added dropwise tothe pellet and allowed to incubate for 7 minutes at room temperature.The mixture was centrifuged again and the pellet of synchronous ringswere resuspended in the RPMI medium. Medium was changed 12 hoursafterward. Potential inhibitors were dissolved in DMSO at theappropriate concentrations. One μl of sample (inhibitor) and 49 μl ofRPMI medium without human serum (incomplete medium) were plated per wellin 96 well plates. For the negative control, one μl of DMSO and 49 μl ofincomplete medium was used. For the positive control, 50 μl of Heparinat 10 μg/ml in incomplete medium was used. At 24-27 hours after sorbitoltreatment, synchronous parasites in the trophozoite stage were culturedat 3.0%-4.5% parasitemia at 3% hematocrit and 150 μl of this culture wasaliquoted to each well. Plates were incubated for 26-28 hours at 40degrees Centigrade to allow the parasites to develop schizonts, segmentinto merozoites and attempt invasion. After incubation, the supernatantabove the settled cells was removed. For each well, about 2 μl of thecells was used for a smear and about 3 μl for flow-cytometry measurementof parasitemia.

For each well, about 3 μl cells were fixed via incubation in a culturetube containing 2 ml of 0.01% glutaraldehyde for at least one hour atroom temperature. Tubes were centrifuged at 250 g for 5 minutes,supernatant aspirated and cells resuspended in Dulbeco's PBS to rime offthe glutaraldehyde. Tubes were centrifuged again, PBS aspirated andcells resuspended in 0.05 mg/ml propidium iodide to cause fluorescenceof parasite DNA. Tubes were shielded from light and incubated in thepropidium iodide overnight at room temperature. For each sample, 20,000cells were analyzed on the FACS flow cytometer and the parasitemiadetermined based on the greater intensity of fluorescence of infectedcells. Percent inhibition as reported in FIG. 4 is the ratio of sampleparasitemia to negative control substrated from 100%. The IC₅₀ values asdetermined in this assay for specific inhibitors in accordance with thepresent invention are reported in Table 5.

                  TABLE 5                                                         ______________________________________                                        IC.sub.50 of Inhibitors                                                                        Malaria Whole Parasite                                                        Red Blood Cell                                               Compound         Infectivity Assay                                            ______________________________________                                        148A               6 μM                                                    III43A           1.7 μM                                                    III79A           1.3 μM                                                    III109A          0.5 μM                                                    I93A             5.8 μM                                                    III128A          6.2 μM                                                    III129A          4.0 μM                                                    III41A           5.0 μM                                                    III45A           5.2 μM                                                    III103A          6.5 μM                                                    87A              3.1 μM                                                    III111A          3.8 μM                                                    ______________________________________                                    

Example 17

Inhibition of critical cysteine proteases from other parasites

Stock solutions (25 μM) of the protease substrate, N-CBZ-Phe-Arg-7-AMC,and 10 mM of inhibitors were prepared in DMSO. A stock solution (1M)dithiothreitol was prepared in 10 mM sodium acetate, pH 6. Proteaseassays contained per reaction a constant amount of: 27 μg purifiedprotease, 3.3 mM DDT, and 33 μM protease substrate. The concentrationrange of protease inhibitors used was 10 nM to 100 μM. Sodium acetatebuffer (100 mM, pH 5.5) was added to a final volume of 1.5 ml perreaction. The variation in the absorbance was measured at 460 nm in aspectrofluorometer. The results are reported in Table 6.

                  TABLE 6                                                         ______________________________________                                        IC.sub.50 of Inhibitors Against Other Parasite                                Cysteine Proteases and Human Cathepsin B                                              T. cruzi    S. mansoni  Human                                         Compound                                                                              Cruzain     Hemoglobinase                                                                             Cathepsin B                                   ______________________________________                                        148A    13 μM    No inhib.   No inhib.                                     III43A  9.0 μM   9.3 μM   No inhib.                                     III79A  24 μM    7.6 μM   No inhib.                                     III109A 21 μM    2.9 μM   No inhib.                                     ______________________________________                                    

What is claimed is:
 1. A method for treating a patient infected with ametazoan parasite, said method comprising administering an amounteffective to kill the parasite of at least one metazoan proteaseinhibitor of general formula

    A-X-B

wherein A is a substituted or unsubstituted homoaromatic ring systemcomprising one to three rings which binds to the S2 subsite of themetazoan protease; B is a substituted or unsubstituted homoaromatic ringsystem comprising one to three rings which binds to the S1 or S1'subsite of the metazoan protease; X is a linker comprising asubstantially planar linear array with a backbone of four to eight atomsin length; wherein A and B are not heterocycles, and X is selected fromthe group consisting of: ##STR22## wherein R is hydrogen or lower alkyl;##STR23## wherein R is hydrogen or lower alkyl; ##STR24## wherein R ishydrogen or lower alkyl, R' is hydrogen, lower alkyl, or aryl, and Y isO or S; ##STR25## wherein R is hydrogen or lower alkyl, R' is hydrogen,lower alkyl, or aryl, and Y is O or S; ##STR26## wherein R is hydrogenor lower alkyl and R' is hydrogen, lower alkyl, or aryl; ##STR27##wherein R and R" are independently selected from hydrogen and loweralkyl; and R' is independently selected from hydrogen, lower alkyl, andaryl; ##STR28## wherein the cyclohexyl group is unsubstituted orsubstituted; and ##STR29## wherein R is independently selected fromhydrogen and lower alkyl; and R' is independently selected fromhydrogen, lower alkyl, and aryl.
 2. A method according to claim 1,wherein the metazoan parasite is selected from the group consisting ofPlasmodium falciparum, Schistosoma mansoni, Trypanosoma cruzi, Giardialamblia, Entoemeba histolytica, Cryptospiridium spp., Leishmania spp.,Brugia spp., Wuchereria spp., Onchocerca spp., Strongyloides spp.,Coccidia, Haemanchus spp., Ostertagia spp., Trichomonas spp.,Dirofilaria spp., Toxocara spp., Naegleria spp., Pneumocystis carinii,Ascaris spp., other Trypanosoma spp., other Schistosome spp., otherPlasmodium spp., Babesia spp., Theileria spp., Anisakis and Isosporabeli.
 3. A method according to claim 1, wherein the metazoan proteaseinhibitor is administered at a dosage of about 0.01 to about 10 μM perkilogram of body weight of patient per day.
 4. A method according toclaim 3, wherein the metazoan protease inhibitor is administered at adosage of about 0.01 to about 1 μM per kilogram of body weight ofpatient per day.
 5. A method according to claim 1, wherein A is selectedfrom the group consisting of phenyl, 1-naphthyl, 1-isoquinolyl,1-phthalazinyl, 3-coumarinyl, 9-phenanthryl and 1-quinolyl.
 6. A methodaccording to claim 1, wherein B is selected from the group consisting ofphenyl, 1-naphthyl, 2-naphthyl, 1-isoquinolyl, 1-phthalazinyl,3-coumarinyl, 9-phenanthryl, 1-quinolyl, 2-quinolyl, 3-quinolyl,6-coumarinyl and 2-chromonyl.
 7. A method according to claim 1, whereinat least one of A, B and X is substituted by at least one substituentselected from the group consisting of hydroxyl, lower alkyl, loweralkoxy, amino, mono-and di-(lower-alkyl)-amino, --NO₂, halogen, aryl,aryloxy, --COOH and --COOR', wherein R' is hydrogen, lower alkyl oraryl.
 8. A method according to claim 1, wherein A is unsubstituted orsubstituted 1-naphthyl and B is unsubstituted or substituted 2-naphthylor phenyl.
 9. A method according to claim 1, wherein X is ##STR30## inwhich R is hydrogen or lower alkyl, R' is hydrogen or lower alkyl and Yis O or S.
 10. A composition for treating a patient infected with ametazoan parasite, said composition comprising a suitable carrier orexcipient and an amount effective to kill the parasite of at least onemetazoan protease inhibitor of general formula

    A-X-B

wherein A is a substituted or unsubstituted homoaromatic ring systemcomprising one to three rings which binds to the S2 subsite of themetazoan protease; B is a substituted or unsubstituted homoaromatic ringsystem comprising one to three rings which binds to the S1 or S1'subsite of the metazoan protease; X is a linker comprising asubstantially planar linear array with a backbone of four to eight atomsin length; wherein A and B are not heterocycles, and X is selected fromthe group consisting of: ##STR31## wherein R is hydrogen or lower alkyl;##STR32## wherein R is hydrogen or lower alkyl; ##STR33## wherein R ishydrogen or lower alkyl, R' is hydrogen, lower alkyl, or aryl, and Y isO or S; ##STR34## wherein R is hydrogen or lower alkyl, R' is hydrogen,lower alkyl, or aryl, and Y is O or S; ##STR35## wherein R is hydrogenor lower alkyl and R' is hydrogen, lower alkyl, or aryl; ##STR36##wherein R' and R" are independently selected from hydrogen and loweralkyl; and R' is independently selected from hydrogen, lower alkyl, andaryl; ##STR37## wherein the cyclohexyl group is unsubstituted orsubstituted; and ##STR38## wherein R is independently selected fromhydrogen and lower alkyl; and R' is independently selected fromhydrogen, lower alkyl, and aryl.
 11. A composition according to claim10, wherein the metazoan parasite is selected from the group consistingof Plasmodium falciparum, Schistosoma mansoni, Trypanosoma cruzi,Giardia lamblia, Entoemeba histolytica, Cryptospiridiurn spp.,Leishmania spp., Brugia spp., Wuchereria spp., Onchocerca spp.,Strongyloides spp., Coccidia, Haemanchus spp., Ostertagia spp.,Trichomonas spp., Dirofilaria spp., Toxocara spp., Naegleria spp.,Pneumocystis carinii, Ascaris spp., other Trypanosoma spp., otherSchistosome spp., other Plasmodium spp., Babesia spp., Theileria spp.,Anisakis and Isospora beli.
 12. A composition according to claim 10, ina dosage unit form wherein the metazoan protease inhibitor is present inan amount sufficient to provide about 0.01 to about 10 μM per kilogramof body weight of patient per day.
 13. A composition according to claim12, in a dosage unit form wherein the metazoan protease inhibitor ispresent in an amount sufficient to provide about 0.01 to about 1μM perkilogram of body weight of patient per day.
 14. A composition accordingto claim 10, wherein A is selected from the group consisting of phenyl,1-naphthyl, 1-isoquinolyl, 1-phthalazinyl, 3-coumarinyl, 9-phenanthryland 1-quinolyl.
 15. A composition according to claim 10, wherein B isselected from the group consisting of phenyl, 1-naphthyl, 2-naphthyl,1-isoquinolyl, 1-phthalazinyl, 3-coumarinyl, 9-phenanthryl, 1-quinolyl,2-quinolyl, 3-quinolyl, 6-coumarinyl and 2-chromonyl.
 16. A compositionaccording to claim 10, wherein at least one of A, B and X is substitutedby at least one substituent selected from the group consisting ofhydroxyl, lower alkyl, lower alkoxy, amino, mono- anddi-(lower-alkyl)-amino, --NO₂, halogen, aryl, aryloxy, --COOH and--COOR', wherein R' is hydrogen, lower alkyl or aryl.
 17. A compositionaccording to claim 10, wherein A is unsubstituted or substituted1-naphthyl and B is unsubstituted or substituted 2-naphthyl or phenyl.18. A composition according to claim 10, wherein X is ##STR39## in whichR is hydrogen or lower alkyl, R' is hydrogen or lower alkyl and Y is Oor S.